Structural insights into immune escape at killer T cell epitope by SARS-CoV-2 Spike Y453F variants

CD8+ T cell immunity, mediated by human leukocyte antigen (HLA) and T cell receptor (TCR), plays a critical role in conferring immune memory and protection against viral pathogens. The emergence of SARS-CoV-2 variants poses a serious challenge to the efficacy of current vaccines. Whereas numerous SARS-CoV-2 mutations associated with immune escape from CD8+ T cells have been documented, the molecular effects of most mutations on epitope-specific TCR recognition remain largely unexplored. Here, we studied an HLA-A24-restricted NYN epitope (Spike448-456) that elicits broad CD8+ T cell responses in COVID-19 patients characterized by a common TCR repertoire. Four natural mutations, N450K, L452Q, L452R, and Y453F, arose within the NYN epitope and have been transmitted in certain viral lineages. Our findings indicate that these mutations have minimal impact on the epitope's presentation by cell surface HLA, yet they diminish the affinities of their respective peptide-HLA complexes (pHLAs) for NYN peptide-specific TCRs, particularly L452R and Y453F. Furthermore, we determined the crystal structure of HLA-A24 loaded with the Y453F peptide (NYNYLFRLF), and subsequently a ternary structure of the public TCRNYN-I complexed to the original NYN-HLA-A24 (NYNYLYRLF). Our structural analysis unveiled that despite competent presentation by HLA, the mutant Y453F peptide failed to establish a stable TCR-pHLA ternary complex due to reduced peptide: TCR contacts. This study supports the idea that cellular immunity restriction is an important driving force behind viral evolution.

and 100 µM were injected over immobilized TCR NYN-I .(lower) Fitting curves for equilibrium binding that resulted in K D s of 19.7 µM and 57.1 µM, respectively.D-E, (upper) N450K-HLA or L452Q-HLA at concentrations of 1. 56, 3.12, 6.25, 12.5, 25.0, 50.0 and 100 µM were injected over immobilized TCR NYN-II .(lower) Fitting curves for equilibrium binding that resulted in K D s of 10.9 µM and 30.6 µM, respectively.Each SPR experiment was conducted with two independent biological replicates, and the repeated results were shown in Figure S3.
Hydrogen bonds and salt bridges were calculated using the Protein Interfaces, Surface, and Assemblies (PISA) server with a cutoff distance of 4.0 Å.The unique bonds for WT and Y453F peptides are highlighted in bold black.Table S4.Interactions between TCR NYN-I and HLA-A24.
Hydrogen bonds, salt bridges and Van der Waals contacts were calculated using the Protein Interfaces, Surface, and Assemblies (PISA) server and CCP4 program with a cutoff distance of 4.0 Å.Table S5.Interactions between TCR NYN-I and NYN peptide.
Hydrogen bonds and Van der Waals contacts were calculated using the Protein Interfaces, Surface, and Assemblies (PISA) server and CCP4 program with a cutoff distance of 4.0 Å.

Figure S2 .
Figure S2.Detection of the decreased TCR: pMHC interaction due to mutations.A, ELISA affinity diagrams of five NYN-TCRs with WT or mutant pHLAs.Each experiment was performed in technical triplicates.The data were shown as the mean ± SEM.Statistical significance was calculated using a twotailed unpaired Student's t test.B-C, (upper) N450K-HLA or L452Q-HLA at concentrations of 1.56, 3.12, 6.25, 12.5, 25, 50 and 100 µM were injected over immobilized TCR NYN-I .(lower) Fitting curves for equilibrium binding that resulted in K D s of 19.7 µM and 57.1 µM, respectively.D-E, (upper) N450K-HLA or L452Q-HLA at concentrations of 1.56, 3.12, 6.25, 12.5, 25.0, 50.0 and 100 µM were injected over immobilized TCRNYN-II .(lower) Fitting curves for equilibrium binding that resulted in K D s of 10.9 µM and 30.6 µM, respectively.Each SPR experiment was conducted with two independent biological replicates, and the repeated results were shown in FigureS3.

Figure S3 .Figure S4 .
Figure S3.The repeated results of SPR experiments.A-J, WT or mutant pHLA proteins at concentrations of 1.56, 3.12, 6.25, 12.5, 25, 50 and 100 µM were injected over immobilized TCRNYN-I or TCR NYN-II .Each SPR experiment was conducted with two biological replicates, with the other replicate shown in Figure2 (A-F) and FigureS2 (B-D).

Figure S5 .
Figure S5.Supplementary figures for the crystal structure of TCR NYN-I -NYN-HLA-A24 complex.A, electron density (2Fo-Fc) of the NYN-peptide (NYNYLYRLF) in the crystal structure of TCR NYN-I -NYN-HLA-A24 complex.Carbon atoms of the peptides are cyan; nitrogen atoms are blue; oxygen atoms are red.HLA-A24 helix is grey.B, electron density (2Fo-Fc) at the interface between TCR NYN-I and HLA-A24.C, electron density (2Fo-Fc) at the interface between TCR NYN-I and the peptide.D, sequence alignment of TCR residues at key positions for the peptide: TCR interactions.E, peptide-binding structure of L452R-HLA predicted by AlphaFold2.The L452R peptide bind HLA-A24 in a conventional direction with side chains of P2-Tyr and P9-Phe accommodated in pockets B and F, respectively.HLA-A24 is shown in grey surface.F, structural superimposition of L452R-HLA-A24 and TCR NYN-I -NYN-HLA-A24 complex.The original and L452R peptide are cyan and light blue, respectively.The mutation site of P6-Arg is highlighted in orange.P4-Tyr and P5-Arg of the peptide formed two steric clashes with CDR3α and CDR1α, respectively.

Table S1 .
SPR summary of pHLAs binding to TCRNYN-Ⅰor TCRNYN-Ⅱ.R work = S h ||F obs (h)|-|F calc (h)|| / S h |F obs (h)|, where F obs (h) and F calc (h) are the observed and calculated structure factors, respectively.No I/s cutoff was applied.‡ R free is the R-value obtained for a test set of reflections consisting of a randomly selected 5% subset of the data set excluded from refinement.* Values from Molprobity server (http://molprobity.biochem.duke.edu/).
NA: not available.*:no measurable binding was observed.Values in the parentheses correspond to the last resolution shell.¶ R meas = S h (n/n-1) 1/2 S i |I i (h) -<I(h)> | / S h S i I i (h), where I i (h) and <I(h)> are the ith and mean measurement of the intensity of reflection h.† *

Table S2 .
X-ray data collection and refinement statistics.